Analyzing Semi-Volatiles by GC-MS Jonathan A. Karty July 31, 2008 Overview GC-MS Instrumentation Description GC-MS Method Sections In Detail Sequence Programming Data Analysis Tips and Tricks 1
Quick Description Agilent 7683B autosampler and tray Can handle up to 100 samples at a time 0.2-10 ul injection volumes 2 different wash solvents can be used Agilent 6890n Gas Chromatograph He carrier gas Split/splitless injector 500:1 split ratio or higher -60 C to +450 C temperatures possible @ 20 C/min Liquid CO 2 cryo option available Agilent 5973 inert mass selective detector EI source (default) or CI source available 10-800 m/z range Positive and negative ions (CI mode) can be analyzed All instrument functions controlled by MSD Chemstation 1701D.02 software GC-MS Availability The undergraduate labs paid half the cost for the new instrument Thus, A315 and A314 lab sections have absolute priority for instrument time A315 uses GC-MS 2 or 3 afternoons throughout the fall semester Their runs last from 2:00 pm 10:00 pm A314 uses GC-MS for 2 weeks in spring semester Check the sign on the GC-MS for their schedule 2
Sample Guidelines Sample needs to be volatile or semi-volatile All analytes MUST pass through a GC column Good rule of thumb, under 350 Da Salts will most likely not work Extremely volatile samples (e.g. CFCs) work great Sample should be 1-20 mg/l More dilute sample can use splitless injection If possible, use a volatile solvent Ether, chloroform, methylene chloride, hexane, ethanol, or something more volatile Non-volatile solvents (e.g. DMF) can be used, but are not preferred If possible, remove non-volatile components Non-volatiles foul up inlet liner and first few cm of GC column GC-MS Overview GC-MS is really two experiments in one Gas chromatography separates compounds by hydrophobicity/volatility Mass spectrometry separates compounds by mass/charge One can utilize the advantages of both techniques in a single analysis However one must make sure sample is compatible with both GC and EI-MS 3
Gas Chromatography Volatile molecules equilibrate between stationary phase and carrier gas M ads M vap Carrier gas: He Default column: 30 m long x 0.25 mm i.d. DB-5MS 95% dimethlysiloxane, 5% phenylsiloxane, 0.25 µm df Affinity for stationary phase favors adsorption, slowing transit through column Temperature increase favors volatilization, accelerating transit through column Oven temperature is the major experimental variable Stationary phase chemistry is another variable Retention time is the measured quantity Other Column Chemistries The MSF owns a handful of columns other than the rather non-polar DB-5 DB-1 (100% dimethylsiloxane, very non-polar) DB-VRX (special volatiles column) CarbonPLOT (carbon stationary phase for permanent gases) DB-17ms (50% phenylsiloxane, 50% dimethylsiloxane, medium polarity) DB-WAX (ethylene glycol, polar phase) Column changeover requires ~2 hr Let MSF staff know 1 day in advance Users may provide their own columns as long as they are stable enough for GC-MS 4
EI-QMF Mass Spectrometry As molecules elute from column, they pass into an electron ionization source 70 ev electrons ionize molecules (M + ) EI often results in extensive fragmentation Ion and fragment m/z ratios analyzed by quadrupole mass filter Only centroid or stick data are recorded Height of centroid peak corresponds to area of raw data peak Full scan takes ~0.3 seconds Default range: 40-500 m/z Scanning wide m/z range can hurt sensitivity (0.22% duty cycle for 40-500 m/z example) SIM mode can increase sensitivity 10-200 fold Policies In Brief All samples should be run as a sequence Users can add more samples during a run All methods and data must initially be stored in the Default directory MSD ChemStation only allows one directory for methods and data to be specified during a sequence Samples are discarded each morning unless we are told in advance 5
GC-MS Method Sections Injector (sets volume to be injected) Inlet (sets split conditions and inlet temp) Column (sets flow rate and column type) Oven (contains temperature program) Aux (sets MS transfer line temperature) Sim/Scan (controls mass spectrometer) Other sections are not routinely used Configure buttons are only for MSF staff A Few Concepts to Remember Inlet temps above 285 C accelerate septum degradation Solvent delay protects the source from high heat capacity solvent peak Relatively large amounts of solvent are introduced during injection CH 2 Cl 2 is 15.6 M with itself; ether is 9.6 M C p and thermal conductivity of solvent vapors differ significantly from He Rapid cooling of EI filament by solvent vapor rapidly degrades it Large amount of solvent ions foul interior of the MS Initial temp should be at least 10 C above bp of solvent, if possible 6
Instrument Control Window This window controls the GC-MS Sequences are controlled from sample tube icons Method is edited from the Oven icon Mass spectrometer is controlled from quadrupole icon A few instrument parameters are monitored in real-time Instrument Control Screenshot 7
GC Method Control Screenshot Injector Tab Injection volume is quantized Washes involve filling the syringe and dispensing it to waste 2 wash solvents available (A and B) One need not use both solvents 4 washes are usually adequate Be sure to use pre and post-injection washes Pumps flush the syringe with the sample Again, 4 pumps are usually adequate 8
Inlets Tab Only use front inlet and He gas Split mode is good for most samples Splitless can be used for dilute or vapor samples Purge flow to split vent should be 30 ml/min @ 1.00 min Try to keep heater to <285 C Gas saver should be set to 15 ml/min 2 minutes after injection All 4 boxes should be checked on this page Pulsed split and pulsed splitless modes are not normally used Columns Tab Make sure installed column in method matches column indicated on sign Always specify: Column: 1 Inlet: Front Detector: MSD Outlest PSI: Vacuum Flow rate should be ~1 ml/min Pressure and velocity are calculated Ramping of flow rate is not commonly used 9
Oven Tab Setpoint is the temperature at start of run C/min is the temperature ramp rate Next is the temperature to be attained at end of ramp step Up to 6 ramp steps can be specified Hold time denotes time spent at Next temp prior to proceeding to next ramp step All the boxes on right should be unchecked Users must NOT change oven configuration Aux Tab Specifies temperature of transfer line between GC oven and EI source Usually set to same temperature as injector port Temperature can be ramped if your analyte is particularly labile 10
GC Method Hints Try to keep runs under 60 minutes Try to include 5-10 minutes at the end of a run @ 250-300 C to clean the column Initial temps below 40 C can take a long time to reach Try to have initial temperature as high as possible for fast run times Inject solvent vapor when making a new method to figure solvent delay If two peaks won t separate, try lowering the temperature ramp Isothermal steps are possible GC Tabs Not Used The tabs for the following GC method parameters are not used Valves Detectors Signals Runtime Options These control hardware we do not own 11
Mass Spectrometry Method Mode (Scan or SIM) Solvent Delay Ensures MS is off when solvent arrives For high BP solvents (e.g. DMF) or volatile analytes, event table can be used to turn MS off during a run EM voltage is always set to 0 (uses value in the tune file) Masses monitored A full range in scan mode (e.g. 40-500) A series of discrete masses in SIM mode Multiple time segments in which different masses are monitored at different times can be employed Real-time window setup Specify time and vertical scale for real-time MS monitor Mass Spectrometry Modes Scan mode Monitors all m/z s in a given range by scanning the quadrupole Must be used if database searching is needed Suffers from low duty cycle SIM mode Specify an m/z and a length of time to monitor it Up to 60 m/z s can be monitored in a group Each m/z can be monitored for up to 100 msec at a time For either mode, try to have scan rate be greater than 3/sec Ensures narrow GC peaks are adequately sampled 12
MS Method Screenshot Programming a Sequence One of the more frustrating aspects of MSD ChemStation in an Open Access lab ChemStation allows 1 directory each for methods and data storage in a sequence Thus, all methods and data are initially stored in the Default/Methods and Default/Data directories Data and methods can be copied to userspecific directories after analysis Cut and paste don t work reliably in the sequence editor 13
General Sequence Instructions If no sequence running, load a method from the Default/Methods directory If no sequence currently running, load sequence blank.s from Default/Sequence directory All analyses are type sample Vial numbers must be 1-100 Data file names must follow WinXP rules Do not add.d (that gets added later by ChemStation) Suggest using email/date-based names like jkarty_jul312008_001 Select a method from the list obtained by double clicking in the method cell Sample name and comment can be left blank All fields to the right of comment are only for automated quantitation experiments Sequence Screenshot 14
If a Sequence is Already Underway Click the Edit button in the box along the bottom of the Instrument Control Window Enter new samples to the sequence then click OK New samples are added the queue Remember, only 1 directory can be specified methods and/or data storage Sequence Helpers Fill and Increment Useful for file names and vial numbers Will increment numbers in successive rows vial numbers 1, 2, 3, 4 File names e.g. jak_001, jak_002, jak_003 Do not use in method column Cannot skip a number Fill and No Increment Useful for method names and sample types Merely copies top entry into all successive cells 15
Finalizing a Sequence Click the OK icon when finished Click the Save Sequence icon Give it a file name based on the date Jul312008_a DO NOT OVERWRITE BLANK.S Click the Validate Sequence icon This is one last chance to make sure you entered everything correctly Be sure Run entire method is checked If something is wrong, click Edit Sequence icon and fix it, the resave and validate Running a Sequence Make sure autosampler wash vials A and B are filled with appropriate solvents 4 ml vials for washing Some have been labeled with solvent names Fill the up to the neck Wash A goes in A11, Wash B goes in B8 on turret Make sure waste vials are in place When everything is OK, click Run Sequence icon Be sure Run Entire Method is checked Check Overwrite existing files at your own risk However, if a file already exists, sequence will stop 16
Common Pop-up Messages Method Resolution for Changed Configuration Usually column or oven max temperature Review the method in the window that pops up Make sure column in the method is the right chemistry and matches column installed on system Override solvent delay Unless you injected solvent vapor to find the solvent delay, ALWAYS ANSWER NO Filename already exists Change filename in sequence, rerun the row in question Avoid this by using Validate Sequence and CAREFULLY checking the report Method Resolution Screen 17
Override Solvent Delay Screen Analyzing Data Once recorded, data are processed in the Data Analysis The basics of using Data Analysis are covered in the document How to access GC-MS data from outside the MSF and ChemStation Tutorial on the MSF website NIST 02 library can be used for database comparisons of EI data Snapshot icon can be used to look at data currently being recorded 18
Data Analysis Icons The chromatogram icon at far left redraws the TIC The m/e icon allows one to create up to six extracted ion chromatograms These plot intensity of a particular ion throughout the run Integration icons are used to compute peak areas Output can be printed either to paper (MSF2420) or pdf (deskpdf) Printer must be specified as Windows default printer PRIOR to submitting the job Data Analysis Screenshot Snapshot icon Chromatogram icons Integration icons 19
Walk-Up Training The GC-MS is so easy to use, training will be done as you bring up samples Make sure Angie or Jon is around to conduct the training Labmates ARE NOT authorized to train each other 20